Development of hybrid plant breeding has made possible considerable advances in quality and quantity of crops produced. Increased yield and combination of desirable characteristics, such as resistance to disease and insects, heat and drought tolerance, along with variations in plant composition are all possible because of hybridization procedures. These procedures frequently rely heavily on providing for a male parent contributing pollen to a female parent to produce the resulting hybrid.
Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinating if pollen from one flower is transferred to the same or another flower of the same plant or a genetically identical plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.
In certain species, such as Brassica campestris, the plant is normally self-sterile and can only be cross-pollinated. In self-pollinating species, such as soybeans and cotton, the male and female plants are anatomically juxtaposed. During natural pollination, the male reproductive organs of a given flower pollinate the female reproductive organs of the same flower.
Maize plants (Zea mays L.) present a unique situation in that they can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. It can self or cross pollinate. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.
The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential. The hybrid progeny of the first generation is designated F1. In the development of hybrids only the F1 hybrid plants are sought. The F1 hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.
Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the male tassel is removed from the growing female inbred parent, which can be planted in various alternating row patterns with the male inbred parent. Consequently, providing that there is sufficient isolation from sources of foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is therefore hybrid (F1) and will form hybrid plants.
Field variation impacting plant development can result in plants tasseling after manual detasseling of the female parent is completed. Or, a female inbred plant tassel may not be completely removed during the detasseling process. In any event, the result is that the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced. Female inbred seed does not exhibit heterosis and therefore is not as productive as F1 seed. In addition, the presence of female inbred seed can represent a germplasm security risk for the company producing the hybrid.
Alternatively, the female inbred can be mechanically detasseled by machine. Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less costly. However, most detasseling machines produce more damage to the plants than hand detasseling. Thus, no form of detasseling is presently entirely satisfactory, and a need continues to exist for alternatives which further reduce production costs and to eliminate self-pollination of the female parent in the production of hybrid seed.
A reliable system of genetic male sterility would provide advantages. The laborious detasseling process can be avoided in some genotypes by using cytoplasmic male-sterile (CMS) inbreds. In the absence of a fertility restorer gene, plants of a CMS inbred are male sterile as a result of factors resulting from the cytoplasmic, as opposed to the nuclear, genome. Thus, this characteristic is inherited exclusively through the female parent in maize plants, since only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred that is not male-sterile. Pollen from the second inbred may or may not contribute genes that make the hybrid plants male-fertile. Usually seed from detasseled normal maize and CMS produced seed of the same hybrid must be blended to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown and to insure cytoplasmic diversity.
One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, et al. However, this form of genetic male sterility requires maintenance of multiple mutant genes at separate locations within the genome and requires a complex marker system to track the genes and make use of the system convenient. Patterson also described a genic system of chromosomal translocations which can be effective, but which are complicated. (See, U.S. Pat. Nos. 3,861,709 and 3,710,511.)
Many other attempts have been made to improve on these systems. For example, Fabijanski, et al., developed several methods of causing male sterility in plants (see EPO 89/3010153.8 publication no. 329,308 and PCT application PCT/CA90/00037 published as WO 90/08828). One method includes delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter. Another involves an antisense system in which a gene critical to fertility is identified and an antisense to the gene inserted in the plant. Fabijanski, et al. also shows several cytotoxic antisense systems. See EP0329308. Still other systems use “repressor” genes which inhibit the expression of another gene critical to male sterility. See PCT/GB90/00102, published as WO 90/08829. For yet another example see U.S. Pat. No. 6,281,348.
A still further improvement of this system is one described at U.S. Pat. No. 5,478,369 in which a method of imparting controllable male sterility is achieved by inactivating or otherwise silencing a gene native to the plant that is critical for male fertility and transforming that plant with the gene critical to male fertility linked to an inducible promoter controlling expression of the gene. That is, the expression of the endogenous sequence is prevented, by any of the methods known to a skilled person in the art for preventing expression of a sequence (such an antisense methods, cosuppression, mutation, use of ribozymes or hairpins, various repression systems and the like, discussed infra.) The plant is thus constitutively sterile, becoming fertile only when the promoter is induced and its linked male fertility gene is expressed.
In a number of circumstances, a male sterility plant trait is expressed by maintenance of a homozygous recessive condition. Difficulties arise in maintaining the homozygous condition, when a restoration gene must be used for maintenance. For example, a natural mutation in a gene critical to male fertility can impart a male sterility phenotype to plants when this mutant allele is in the homozygous state. But because this homozygosity results in male sterility, the homozygous male-sterile line cannot be maintained. Fertility is restored when the non-mutant form of the gene is introduced into the plant. However, this form of line maintenance removes the desired homozygous recessive condition, restores full male fertility in half of the resulting progeny, and prevents maintenance of pure male sterile maternal lines. These issues can be avoided where production of pollen containing the restoration gene is eliminated, thus providing a maintainer plant producing only pollen not containing the restoration gene, and the progeny retain their homozygous condition when fertilized by such pollen. An example of one approach is shown in Dellaporta et al., 6,743,968, in which a plant is produced having a hemizygotic construct comprising a gene that produces a product fatal to a cell, linked with a pollen-specific promoter, and the restoration gene. When crossed with the homozygous recessive male sterile plant, the progeny thus retains the homozygous recessive condition.
As noted, an essential aspect of much of the work underway with male sterility systems is the identification of genes impacting male fertility. Such a gene can be used in a variety of systems to control male fertility including those described herein.
Genetic male sterility results from a mutation, suppression, or other impact to one of the genes critical to a specific step in microsporogenesis, the term applied to the entire process of pollen formation. These genes can be collectively referred to as male fertility genes (or, alternatively, male sterility genes). There are many steps in the overall pathway where gene function impacts fertility. This seems aptly supported by the frequency of genetic male sterility in maize. New alleles of male sterility mutants are uncovered in materials that range from elite inbreds to unadapted populations.
At U.S. Pat. No. 5,478,369 there is described a method by which the Ms45 male fertility gene was tagged and cloned on maize chromosome 9. Previously, there had been described a male sterility gene on chromosome 9, ms2, which had never been cloned and sequenced. It is not allelic to the gene referred to in the '369 patent. See Albertsen, M. and Phillips, R. L., “Developmental Cytology of 13 Genetic Male Sterile Loci in Maize” Canadian Journal of Genetics & Cytology 23:195-208 (January 1981). The only fertility gene cloned before that had been the Arabidopsis gene described at Aarts, et al., supra.
Examples of genes that have been discovered subsequently that are critical to male fertility are numerous and include the Arabidopsis ABORTED MICROSPORES (AMS) gene, Sorensen et al., The Plant Journal (2003) 33(2):413-423); the Arabidopsis MS1 gene (Wilson et al., The Plant Journal (2001) 39(2):170-181); the NEF1 gene (Ariizumi et al., The Plant Journal (2004) 39(2):170-181); Arabidopsis AtGPAT1 gene (Zheng et al., The Plant Cell (2003) 15:1872-1887); the Arabidopsis dde2-2 mutation was shown to be defective in the allene oxide syntase gene (Malek et al., Planta (2002)216:187-192); the Arabidopsis faceless pollen-1 gene (flp1) (Ariizumi et al, Plant Mol. Biol. (2003) 53:107-116); the Arabidopsis MALE MEIOCYTE DEATH1 gene (Yang et al., The Plant Cell (2003) 15: 1281-1295); the tapetum-specific zinc finger gene, TAZ1 (Kapoor et al., The Plant Cell (2002) 14:2353-2367); and the TAPETUM DETERMINANT1 gene (Lan et al, The Plant Cell (2003) 15:2792-2804).
The table below lists a number of known male fertility mutants or genes from Zea mays.
GENE NAMEALTERNATE NAMEREFERENCEms1 male sterile1male sterile1, ms1Singleton, W R and Jones,D F. 1930. J Hered 21: 266-268ms10 male sterile10male sterile10, ms10Beadle, G W. 1932.Genetics 17: 413-431ms11 male sterile11ms11, male sterile11Beadle, G W. 1932.Genetics 17: 413-431ms12 male sterile12ms12, male sterile12Beadle, G W. 1932.Genetics 17: 413-431ms13 male sterile13ms*-6060, male sterile13,Beadle, G W. 1932.ms13Genetics 17: 413-431ms14 male sterile14ms14, male sterile14Beadle, G W. 1932.Genetics 17: 413-431ms17 male sterile17ms17, male sterile17Emerson, R A. 1932.Science 75: 566ms2 male sterile2male sterile2, ms2Eyster, W H. 1931. J Hered22: 99-102ms20 male sterile20ms20, male sterile20Eyster, W H. 1934. Geneticsof Zea mays. BibliographiaGenetica 11: 187-392ms23 male sterile23ms*-6059, ms*-6031, ms*-West, D P and Albertsen,6027, ms*-6018, ms*-6011,M C. 1985. MNL 59: 87ms35, male sterile23, ms*-Bear7, ms23ms24 male sterile24ms24, male sterile24West, D P and Albertsen,M C. 1985. MNL 59: 87ms25 male sterile25ms*-6065, ms*-6057,Loukides, C A; Broadwater,ms25, male sterile25, ms*-A H; Bedinger, P A. 1995.6022Am J Bot 82: 1017-1023ms27 male sterile27ms27, male sterile27Albertsen, M C. 1996. MNL70: 30-31ms28 male sterile28ms28, male sterile28Golubovskaya, I N. 1979.MNL 53: 66-70ms29 male sterile29male sterile29, ms*-JH84A,Trimnell, M R et al. 1998.ms29MNL 72: 37-38ms3 male sterile3Group 3, ms3, male sterile3Eyster, W H. 1931. J Hered22: 99-102ms30 male sterile30ms30, msx, ms*-6028, ms*-Albertsen, M C et al. 1999.Li89, male sterile30, ms*-MNL 73: 48LI89ms31 male sterile31ms*-CG889D, ms31, maleTrimnell, M R et al. 1998.sterile31MNL 72: 38ms32 male sterile32male sterile32, ms32Trimnell, M R et al. 1999.MNL 73: 48-49ms33 male sterile33ms*-6054, ms*-6024,Patterson, E B. 1995. MNLms33, ms*-GC89A, ms*-69: 126-1286029, male sterile6019,Group 7, ms*-6038, ms*-Stan1, ms*-6041, ms*-6019, male sterile33ms34 male sterile34Group 1, ms*-6014, ms*-Patterson, E B. 1995. MNL6010, male sterile34, ms34,69: 126-128ms*-6013, ms*-6004, malesterile6004ms36 male sterile36male sterile 36, ms*-MS85A,Trimnell, M R et al. 1999.ms36MNL 73: 49-50ms37 male sterile37ms*-SB177, ms37, maleTrimnell, M R et al. 1999.sterile 37MNL 73: 48ms38 male sterile38ms30, ms38, ms*-WL87A,Albertsen, M C et al. 1996.male sterile38MNL 70: 30ms43 male sterile43ms43, male sterile43, ms29Golubovskaya, I N. 1979. IntRev Cytol 58: 247-290ms45 male sterile45Group 6, male sterile45,Albertsen, M C; Fox, T W;ms*-6006, ms*-6040, ms*-Trimnell, M R. 1993. ProcBS1, ms*-BS2, ms*-BS3,Annu Corn Sorghum Indms45, ms45′-9301Res Conf 48: 224-233ms48 male sterile48male sterile48, ms*-6049,Trimnell, M et al. 2002.ms48MNL 76: 38ms5 male sterile5: ms*-6061, ms*-6048, ms*-Beadle, G W. 1932.6062, male sterile5, ms5Genetics 17: 413-431ms50 male sterile50ms50, male sterile50, ms*-Trimnell, M et al. 2002.6055, ms*-6026MNL 76: 39ms7 male sterile7ms7, male sterile7Beadle, G W. 1932.Genetics 17: 413-431ms8 male sterile8male sterile8, ms8Beadle, G W. 1932.Genetics 17: 413-431ms9 male sterile9Group 5, male sterile9, ms9Beadle, G W. 1932.Genetics 17: 413-431ms49 male sterile49ms*-MB92, ms49, maleTrimnell, M et al. 2002.sterile49MNL 76: 38-39
There remains a need to identify nucleotide sequences critical to male fertility in plants. There also remains a need to identify regulatory regions which preferentially direct expression to male tissue of a plant.
In the present invention the inventors provide novel DNA molecules and the amino acid sequence encoded that are critical to male fertility in plants. These can be used in any of the systems where control of fertility is useful, including those described above.
Thus, one object of the invention is to provide a nucleic acid sequence, the expression of which is critical to male fertility in plants and in which a mutation of the sequence causes male sterility when in the homozygous state.
Another object is to provide regulatory regions that preferentially direct expression of operably linked nucleotide sequences to male tissue(s) of a plant.
A further object of the invention is to provide a method of using such nucleotide sequences to mediate male fertility in plants.
Further objects of the invention will become apparent in the description and claims that follow.